Optical assays are a powerful tool in cellular electrophysiology. However, currently-available approaches have not reached their full potential. The major limitation of existing optical systems is that they are unable to determine the absolute voltage in the cell. Current commercial approaches report only qualitative relative changes in voltage, & there is no information on absolute resting potential, diastolic potential, or action potential amplitude. Prior research attempts to develop absolute voltage reports have been unsuccessful. In addition, currently available voltage- sensitive dyes (VSDs) offer a very limited experimental duration (typically < 30 min) dyes due to: 1) high washout & internalization rates, which removes them from the electrically active cell membrane; 2) high photo-toxicity, which reduces the possible exposure time for measurements; & 3) acute dye toxicity, which limits membrane loading & illumination, resulting in small signals & low signal to noise ratio. This proposal overcomes these 2 major obstacles to develop & optimize our novel system which combines our VSDs & our unique & robust optical & analytical system which determines absolute membrane potential. Our integrated quantitative Optical Electrophysiology (qOEP) system consists of our patented long lasting VSDs, optimized experimental protocols, optical detection system, & analytical software. Our VSDs, which operate in the red/NIR spectral range, have reduced acute chemical & photo-toxicity, increased sensitivity, & slower washout/ internalization rate. This gives them the ability to be used in experiments up to 4 hours. This dramatic improvement revolutionizes the types of experiment which can be performed. Specifically, slower internalization rate gives the experimenter time to calibrate the VSD, so that the measured light intensity can be directly correlated with transmembrane potential. The spectral properties & stability of this new generation of VSDs has been combined with advances in electronics & circuitry that increase signal sensitivity & allow for qOEP. Dye performance & signal processing are species & organ/cell type-specific. These systems have high degrees of cellular heterogeneity & connective tissue relative to cultured cells. To develop a consistent system we will optimize our qOEP system specifically for work with electrically syncytial preparations of induced pluripotent stem cell derived (IPSCD) cardiac myocytes. The goal is to optimize a cell system (stem cell derived cardiac myocytes) & the dyes to make an integrated optical system that makes qOEP available to almost any lab. This transformation will be similar to the way that the advent of molecular biology kits made complex molecular biological techniques accessible to all. The long term commercial opportunity is in cardiac safety screening to determine the arrhythmogenic potential of new drug candidates in stem cell derived cardiac myocytes. Our novel system has the potential to have significant impact in both the financial & human health aspects of drug development. Successful completion of Phase I will result in a system consisting of sensors, dyes, illumination sources, & software that can be used for beta testing in Phase II. In Phase II we will develop software, support, packaging & optimized hardware for a turn-key commercial system.